The goal of this program is to design a hammerhead ribozyme having therapeutic activity against HIV-1. The present proposal, which focuses on developing a ribozyme directed against tat mRNA, addresses these aspects: intracellular stability of the ribozyme, target selection, and optimized ribozyme catalytic activity. Stability in human cells will be investigated by measuring the effects of defined terminal modifications on the decay kinetics of an anti-tat ribozyme, and the effects of the various modifications on ribozyme activity will be assessed in vitro and in vivo. Target selection, which is important because there are many possible choices that are likely to vary widely in effectiveness, will focus on target accessibility and sensitivity of various target triplets to cleavage. This aspect will be studied primarily in vitro, although the conclusions will be confirmed in vivo. Improvements in anti-tat ribozyme activity will be addressed by introducing imperfections into ribozyme- target base pairing in order to increase the rate of cleavage. Since there are so many possible imperfections, theoretical considerations will be used to identify ribozymes expected to be optimally active. These will then be examined in vitro. In addition, a bacterial system will be used to screen for exceptionally active mutant ribozymes. Results from the biochemical and bacterial studies of mispairing will constrain the theory, and from the combination will emerge ribozymes to be tested in human cells for anti-HIV activity. It is expected that the net result of the study will be a very stable, highly active ribozyme directed at the most accessible and cleavable site in tat RNA. This ribozyme will be tested for its ability to block HIV-1 replication in cultured cells. Since kinetic parameters of ribozyme-target interactions will be examined in vitro for all of the modifications introduced, it should be possible to attribute the effects of the modifications to particular aspects of a proposed reaction scheme. This information may contribute to a better understanding of the role played by particular regions of the nucleotide sequence in ribozyme action.